1

Workflow of the Manual Purification of N/NC5-enriched proteins

Karishma G. ShettyOctober 7th, 2005

2

Criteria for manual purification

• If the extinction coefficient is less than 4000.

• If the protein had previously failed purification in the AKTA Express

3

Day One

Two Step Purification

Day Two

SDS-PAGE of fractions

collected by AKTA

Day Three

Pool desired fractionsand

Concentrate

Day Five

Final SDS-PAGEand

Mass Spec

Day Six

Bulk Uploadand

NMR Sample Prep

Day Four

Buffer Exchangeand

Concentrate

Outline

4

1Obtain necessary info from

Spine for each protein

1Obtain necessary info from

Spine for each protein

4Sonicate cell suspension

(Total)

4Sonicate cell suspension

(Total)

Day One

6Filter supernatant (0.45m)

(Soluble)

6Filter supernatant (0.45m)

(Soluble)3

Resuspend pellet in 25 ml of Lysis Buffer

3Resuspend pellet in 25 ml of Lysis Buffer

2Get pellet from

freezer

5Obtain supernatant by

centrifugation

7Prepare Ni-NTA

Superflow column

8Pour the supernatant into

The Ni-NTA Superflow Column (F.T.)

9Wash the Column with

50ml of Wash Buffer(W)

9Wash the Column with

50ml of Wash Buffer(W)

10Elute the protein from the

Ni-NTA column using10ml of Elution Buffer (E)

10Elute the protein from the

Ni-NTA column using10ml of Elution Buffer (E)

11Run an SDS PAGE Gel Of the Total, Soluble,

Flow through, Wash andElution Samples

11Run an SDS PAGE Gel Of the Total, Soluble,

Flow through, Wash andElution Samples

12Inject the Samples into

the AKTA Express and let them run overnight .

12Inject the Samples into

the AKTA Express and let them run overnight .

5

SDS Page

6

Day Two

Analyze chromatographand decide which fractions to run

for SDS-PAGE

Decide which fractions to pool based on result of chromatograph and SDS-PAGE

MAINTAIN THE AKTA

7

Day Three

Pool fractions Based Unicorn

Result

Pool fractions Based Unicorn

Result

Concentrate Sample to 0.5-1.0 mMBy AmiconUltrafree

Device

Concentrate Sample to 0.5-1.0 mMBy AmiconUltrafree

Device

Determine Concentration at 280nm by diluting protein with 6M Guanadine + 10mM Tris, pH 7.5

In case of precipitation

Stop further concentrating,remove precipitate bycentrifugation andAnalyze supernatant

8

Day Four

Buffer exchange into selected NMR Buffers using desalting

columns if there is enough protein.

Concentrate Sample to 0.5-1.0 mM by Amicon Ultrafree Device

Concentrate Sample to 0.5-1.0 mM by Amicon Ultrafree Device

Upload purification record to SPiNE and obtain PST IDs

Bulk Purification Uploads into SPiNE – Purification Template

SR361.002

9

Day Five

Final SDS-PAGE

GelFor Purity

Mass Spec To

Confirm MW

Proteins with MW with adeviation greater than 500 Daltons from the reported value in SPiNE are held and submitted for LC-MS analysis (PeterLobel’s group).

10

Day Six

SDS-PAGEpictures

taken

InformationPlaced in SPINsIn JPEG format

InformationUploaded ontoSPiNE

NMR Samples Made, placed in the inbox and email sent out (Swapna, Tom and Rong)

Protein information uploaded into SPINS from SPiNE.

Upload PSTInformationinto SPiNE

11

Proteins attemptedto be purified

147 ProteinsSuccessfully purified

Purification Flowchart

Failed purification

42 Proteins (28.6%) 105 Proteins (71.4%)

E x S <= 4 E x S > 4

10 Proteins (23.8%)32 Proteins (76.2%)

No Binding or Aggregates in GF

Change the buffer & purifyManually or make a new Construct.NOTE: Test for hex-his tag.

12

Proteins attemptedto be purified

147 ProteinsSuccessfully purified

Purification Flowchart

Failed purification

42 Proteins (28.6%) 105 Proteins (71.4%)

E x S <= 8 E x S > 8

7 Proteins (16.67%)35 Proteins (83.33%)

Change the buffer & purifyManually or make a new Construct.NOTE: Test for hex-his tag.

No Binding or Aggregates in GF